Development of a real-time NASBA for human metapneumovirus
Abstract number: o65
Bufflier E., Savelli H., Jacobs F., Moore C., Van de Wiel P.
The aim was to develop and evaluate human Metapneumovirus (hMPV) reagents based on NASBA amplification and real-time detection with molecular beacons.
hMPV RNA is isolated using a semi automated magnetic extraction method (NucliSens® miniMag). An internal control is added to the sample prior to nucleic acid extraction. Primers are directed against the M-gene region of the hMPV genome (subtypes A1, A2, B1 and B2), and against the internal control molecule. A FAM-labelled molecular beacon probe is used to detect hMPV amplicons, while a ROX-labelled molecular beacon is designed to detect the internal control amplicons, amplified in the same reaction. Amplification and real-time detection reactions are performed in a NucliSens® EasyQ Analyser. The extraction and amplification-detection reactions to detect hMPV in nasal swab samples can be performed within approximately 3.0 hours.
The analytical sensitivity of the NASBA reaction was determined to be close to 10 copies per input (90% hit rate) using serial dilutions of in vitro hMPV RNA in direct amplification, while in combination with extraction, the sensitivity was found to be 263 copies per input (90% hit rate). No cross reactivity was observed with PIV 1, PIV 2, PIV 3, PIV 4, RSV A, RSV B, Sars CoV, Influenza A and Influenza B. The NASBA real time reagents to detect both hMPV and RSV were further investigated in a clinical study performed in Cardiff in 20042005. Amongst 1158 clinical respiratory samples tested (Nasopharyngeal Swabs, Nasal Swabs, Throat Swabs and Nasopharyngeal Aspirates) the NASBA allowed to detect 22 hMPV positive samples (ca. 2%). Finally, the efficiency of the method to detect the four A1, A2, B1 and B2 subtypes of hMPV was demonstrated on viral stocks.
The data showed that the real-time hMPV reagents allows a rapid and sensitive qualitative detection of hMPV. The use of standardized reagents offers considerable benefits in a routine setting for the clinical management of patients with hMPV infections.
|Session name:||XXIst ISTH Congress|
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